Summary of Work: Improved understanding of the structure of HIV proteins can be useful in unraveling the function of the proteins and in understanding the mechanisms of the function of these proteins. This structural knowledge can also be useful in designing novel anti-HIV agents. X-Ray crystallography is the most accurate technique for determination of protein structures. Major drawbacks of the technique, however, are that it can be very time consuming and is dependent on the availability of protein crystals. Molecular modeling programs, on the other hand, can provide insights into the protein structure based on, amongst other things, homology with proteins whose structures are known. Although this approach is attractive, the results have often been less reliable than desired because insufficient information about the protein is available or the degree of homology with proteins of known structure is less than needed for the development of an accurate model. If information such as which amino acid residues are on the surface of the protein is available, however, the quality of the computer generated model can be significantly increased. This project is designed to probe the tertiary structure of HIV proteins using a combination of chemical modifications, enzymatic degradations and mass spectrometric identification. Recombinant HIV p24 was chosen as the first protein to examine, both because we were working with this protein in other projects (epitope mapping) and because the structure was not known. The first chemical reaction we used to probe the surface of the protein was glycinamidation of aspartic and glutamic acid residues. This reaction was carried out under non-denaturing conditions for one hour at the picomole level. The short reaction time was used to ensure that only partial reaction (most exposed residues) would occur. After the reaction was stopped, the resulting modified protein was digested with chymotrypsin. MALDI/MS analysis showed acidic residues at positions 188, 94, 172, 111, 165, and 176 were modified. Acetylation of lysine residues has also been carried out. Relative reactivities of five lysines have been determined, (positions 70, 131, 140, 170 and 158) while the relative reactivities of six other lysines have not been determined as yet. A preliminary molecular structure was modeled using inverse folding algorithms to identify structures possessing folds compatible with p24. The identified folds, those belonging to the structure of he cytokine proteins interleukin 4 and interleukin 5, were used as a basis and point of departure for developing a model of the HIV p24 gag protein which also incorporates the surface residues identified in this project and the epitope determined in our epitope mapping project. At this point the crystal structure of the N-terminal part of the molecule was reported. The structure reported did not encompass the C-terminal which is essential to viral assembly. The model proposed was quite similar to the reported structure. Incorporation of the surface accessibility results from our study along with the N-terminal structure will permit us to improve the model of the C-terminal domain.